The over arching hypothesis is that the brain is an important reservoir for retroviruses because of their ability to infect long lived terminally differentiated cells and viral products released from these cells can cause immune activation and neuronal injury Aim 1: To understand the mechanism of viral persistence in brain If there is any hope to eradicate HIV, close attention to the viral reservoirs in the brain is necessary. They brain is a unique site of viral latency since it infects resident macrophages/microglia and astrocytes. These cells have very low turnover rate, and the mechanism of viral entry and persistence is very different than that of lymphocytes which are the major cell type infected by the virus in the lymphoid organs. Our laboratory has focused on studying the mechanism of viral entry in astrocytes. We have found that while free viral particles can enter these cells, cell to cell contact with lymphocytes is the most efficient way to infect astrocytes. The mechanisms by which T cells facilitate viral entry is being explored. We have also found that upon viral entry, the virus can enter the endolysosomal pathway which acts as a host defense mechanism. Hence strategies than modulate this pathway could have a significant effect on the establishment of a reservoir in the brain. These findings were presented at the Conference on Retroviruses and Opportunistic Infections in March 2012. Aim 2: To investigate the mechanism of neuronal injury by HIV and endogenous retroviruses Despite the use of antiretroviral agents and excellent control of the virus in the periphery, HIV infected patients continue to develop cognitive impairment. Currently available antiretroviral agents have no effect on the production of early viral proteins once the virus has integrated into the chromosome. One of these proteins, Tat, has been shown to be neurotoxic. Our laboratory was one of the first to demonstrate its toxic potential and we are now investigating the mechanisms by which it causes neurotoxicity. We have found that the protein can cause synaptic injury at very low concentrations without causing neuronal death. We have characterized the proteins and the morphological changes at the level of the dendrites in human neurons and are further investigating the underlying mechanisms. We are in the process of developing a very sensitive assay using proximity ligation to measure Tat protein in biological samples from patients with HIV infection. Using a similar approach we are investigating the mechanisms by which the envelop protein of an endogenous retrovirus-K causes neurotoxicity. We have cloned the gene into an expression vector and are now conducting experiments to characterize its effects on neurons in vitro Aim 3: To investigate the mechanism of HIV-induced lymphocyte activation and subsequent neuronal injury Patients treated with antiretroviral drugs may occasionally develop a T cell mediated encephalitis that can be fatal. Our group has characterized this syndrome as the CNS-immune reconstitutions syndrome and we were one of the first to characterize the clinical and pathological features of this entitity. We have also found these similar clinical syndromes can occur in patients with JC virus infection and we have published several papers characterizing the clinical entity. We have reasoned that because of the inability to control the viral replication fully in the CNS reservoirs, the lymphocytes traffic to the brain along an antigenic gradient. We have shown that the Tat protein can activate lymphocytes in vitro and these activated T cells can cause significant neurotoxicity by the release of granzymes. We have found that the Tat protein enters T cells and then activates these cells in a NF-kB dependent manner. We have also shown that the activated T cells can be detrimental to neural progenitor cells and prevent their differentiation. This likely contributes to permanent loss of neurons. We will now continue to explore the mechanisms of T cell activation and the subcellular mechanisms by which granzyme B causes neurotoxicity In summary, we have shown that astrocytes in the brain are an important reservoir for HIV and that cell to cell contact with lymphocytes is necessary for viral entry and the lysosomal pathway in these cells regulates the intracellular trafficking of the virus and its ultimate ability to successfully infect these cells. Further, we have shown that the HIV protein Tat and the env protein of endogenous retrovirus-K are neurotoxic and we are now the underlying mechanisms involved in these effects. Finally, we have also discovered that the Tat protein of HIV can stimulate T cells in a T cell receptor independent manner using a unique mechanism. These activated T cells cause neuronal injury via the release of granzyme B that acts on cell receptors causing a cascade of events leading to neuronal dysfunction.